The Preparation And Evaluation Of Pei-Functionalized Rice Bran Polysaccharide And Carbon Nanotubes Gene Delivery Vectors

Efficient and safe gene delivery vectors are the key to the success of gene therapy.Non-viral vectors show many better profiles,such as lower immunogenicity and lower production,than viral ones.However,it was the flip side of these vectors,potent cytotoxicity and low transfection efficiency,that limited their application.Natural macromolecular polysaccharides and inorganic nanomaterials have become hotspots in gene carrier research due to low cytotoxicity.However,both types of vectors need to be modified with a positively charged compound to achieve higher transfection efficiency than traditional gene delivery vehicles.In this paper,rice bran polysaccharides(RBP)and carbon nanotube(CNT)were selected as the research objects.The properties of the modified materials as gene carriers were evaluated by modifying polyethyleneimine.As follows are the main details:PEI-modified rice bran polysaccharide(RBP/PEI30K)was prepared by the condensation of hydroxyl groups on rice bran polysaccharide with amino groups on PEI molecule.PEI-modified carbon nanotubes(CNT/PDA/PEI30K)were derived from CNT/PDA which was synthesized by carbon nanotube-coated PDA.The chemical structures of those vectors were confirmed by infrared spectroscopy,and the DNA binding feasibility of the vectors was confirmed by electrophoresis.Elemental analysis and particle size analysis were used to characterize those vectors.The DNA binding form and ability of the vectors was explored by spectral analysis,and the in-vitro release profile of DNA was explored in the vector/DNA complex at different pH conditions.The biosafety of the vectors was evaluated by MTT and RTCA experiments,and its effectiveness was proved by cell uptake and in-vitro transfection experiments.Its transfection efficiency was further improved by the modification of the TAT peptide.Infrared absorption spectroscopy and elemental analysis confirmed that both vectors were successfully synthesized.RBP/PEI30K and CNT/PDA/PEI30K completely agglomerated DNA at the weight ratio of 1.6:1 and 2:1,respectively,and prevented DNA from being hydrolyzed by DNase I.The particle size of the RBP/PEI30K/DNA complex was approximately 150 nm.Nanoparticles,prepared by dispersing CNT/PDA/PEI30K in water,were approximately 40 nm.The results of electrophoresis and spectroscopy showed that the DNA binding form of the two vectors,the RBP/PEI30K/DNA complex and CNT/PDA/PEI30K/DNA,was electrostatic and embedded,and its DNA binding constants were 1.8 x 10~7 and 2.3 x 10~7,respectively.DNA was released from the vector/DNA complex at different pH conditions,and the release efficiency above 80%.Results from the MTT and RTCA experiments indicated that the cytotoxicity of the two vectors was lower than that of PEI30K at the same concentration.The vector/DNA/EB complexes were successfully taken up by HeLa cells and 293T cells.The transfection efficiency of RBP/PEI30K and CNT/PDA/PEI30K on 293T cells was 38.59%and 9.52%,respectively,at the weight ratios of 5:1 and 2.5:1.After the TAT peptide was inserted,the cytotoxicity of RBP/PEI30K/TAT was greater than that of RBP/PEI30K at the same concentration,but lower than that of PEI30K.The cytotoxicity of CNT/PDA/PEI30K/TAT was lower than that of CNT/PDA/PEI30K and PEI30K at the same concentration.The transfection efficiency of RBP/PEI30K/TAT and CNT/PDA/PEI30K/TAT on 293T cells was 43.53%and 34.53%,respectively,at the weight ratios of 0.75:1 and 3:1.Above all,both RBP and CNT can be used as effective and safe gene vectors.